Arrays of surface-bound binding agents, known in the art as chemical arrays, may be used to detect the presence of particular targets, e.g., biopolymers, in solution. The surface-bound probes may be oligonucleotides, peptides, polypeptides, proteins, antibodies or other molecules capable of binding with target molecules in solution. Such binding interactions are the basis for many of the methods and devices used in a variety of different fields, e.g., genomics (in sequencing by hybridization, SNP detection, differential gene expression analysis, identification of novel genes, gene mapping, finger printing, etc.), CGH, location analysis and proteomics.
One typical array assay method involves biopolymeric probes immobilized in an array on a substrate, such as a glass substrate or the like. A fluid containing sample is placed in contact with the array substrate, covered with another substrate such as a coverslip or the like to form an assay area and placed in an environmentally controlled chamber such as an incubator or the like. Usually, the targets in the sample bind to the complementary probes on the substrate to form a binding complex. The pattern of binding by target molecules to biopolymer probe features or spots on the substrate produces a pattern on the surface of the substrate and provides desired information about the sample. In certain instances, the target molecules are labeled with a detectable tag such as a fluorescent tag or chemiluminescent tag. The resultant binding interaction or complexes of binding pairs are then detected and read or interrogated, for example by optical means, although other methods may also be used. For example, laser light may be used to excite fluorescent tags, generating a signal only in those spots on the biochip that have a target molecule and thus a fluorescent tag bound to a probe molecule. This pattern may then be digitally scanned for computer analysis.
For each pixel of a scan, a light detector (e.g., a photomultiplier tube) typically detects light emitted from the surface of a microarray, and outputs an analog signal that changes in amplitude according to the amount of emitted light entering the detector. This analog signal is usually sampled and digitized using an analog-to-digital converter (A/D converter) and integrated using a signal processor (e.g., a DSP) to provide data, e.g., a numerical evaluation of the brightness of the pixel. This data is usually stored and analyzed at a later date.
Current detection methodologies, however, are limited because the range of light intensity emitted by an array generally exceeds the linear dynamic range of the photodetection systems used for the detection of that light. Accordingly, in scanning an array, typical photodetection systems produce a significant number of data points that are either saturated (i.e., at or above the maximum of the linear dynamic range of the detector), or indistinguishable from background (i.e., at or below the minimum of the dynamic range of the detector).
While the gain of photodetection system may be adjusted (i.e., increased or decreased) in an attempt to maximize both signal strength and detection, such adjustments often have little effect on the overall quality of the data produced by the photodetection system because decreasing the gain of a detection system decreases the sensitivity of the system (i.e., decreases its ability to detect low magnitude signals). On the other hand, increasing the gain of the detection system often causes saturation of high intensity signals. In addition, consecutive scans at different detection gain increases the time per scan, and leads to photobleaching of the fluorescent dyes used in typical array experiments.